Nucleotides are one of the most important cellular metabolites. Nucleotides are found primarily as the monomeric units comprising the major nucleic acids of the cell, RNA and DNA. However, they also are required for numerous other important functions within the cell. These functions include energy stores in phosphate transfer reactions (ATP); as coenzymes (for example, NAD+, NADP+, FAD and coenzyme A); mediators cellular processes (such as cyclic-AMP); allosteric effector on enzyme activity; and activated intermediates (S-adenosylmethionine).
Nucleotide analogues are chemically synthesized and used as therapeutics. Nucleotide analogues can be utilized to inhibit specific enzymatic activities, for example, as antitumor agents that interfere with the synthesis of DNA and thereby preferentially kill rapidly dividing cells such as tumor cells. Some commonly used nucleotide analogues in chemotherapy are 6-mercaptopurine, 5-fluorouracil, 5-iodo-2′-deoxyuridine and 6-thioguanine. Synthesis of DNA is disrupted because the nucleotide analogues prevent correct Watson-Crick base-pairing.
Nucleotide analogues are also used as antiviral agents. Example are abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zalcitabine, and zidovudine. For example, AZT (azidothymidine) and ddI (dideoxyinosine) are use to inhibit replication of HIV. Purine-containing nucleotide analogues are used to treat gout, for example, allopurinol that inhibits the activity of xanthine oxidase, an enzyme involved in de novo purine biosynthesis. Additionally, nucleotide analogues are used to suppress the immune system after organ transplantation and reduce transplant rejection.
Nucleotide analogues, in their phosphorylated form, are also included in small polymeric sequences used as antisense RNA, siRNA (small interfering RNA) or miRNA (micro RNA) to control the transcription and translation of genes related to cancer or viral infections.
Antisense mRNA is an mRNA transcript that is complementary to endogenous mRNA, that is, the noncoding strand complement to the coding strand. A strategy to block expression of a gene of interest is to introduce a transgene coding for antisense mRNA. Analogous molecules with modified backbones using nucleotide analogues have been designed which change various characteristics of antisense RNA, such as instability to degradative enzymes or ability to form stable double strands with the complementary sense RNA. Some alternative antisense molecules include phosphorothioate, morpholino, PNA (peptide nucleic acid), LNA (locked nucleic acid), and 2′-O alkyl oligos.
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long RNA molecules that play a variety of roles. SiRNAs have a well defined structure that consist of a short, usually 21-nt, double-strand of RNA with 2-nt 3′ overhangs on either end. Most notably, siRNA is involved in the RNA interference pathway (RNAi) where the siRNA interferes with the expression of specific genes. In addition, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated. SiRNAs were first discovered as part of post-transcriptional gene silencing (PTGS) in plants (see U.S. Pat. No. 7,056,704 and Hamilton and Baulcombe, Science, 1999, 286, 950-952). Synthetic siRNAs have also been shown to induce RNAi in mammalian cells (see Elbashir et al., Nature, 2001, 411, 494-498) with led to interest in harnessing RNAi for biomedical research and drug development.
Micro RNA (miRNA) are small ribonucleic acid chains, about 22 nt long that are implicated in cell growth and apoptosis, embryonic development, neuronal plasticity and remodeling, and even insulin secretion. An overabundance of miRNA has been reported in cases of Fragile X Mental Retardation while some cancers have been reported to have downregulated miRNA genes.
Antisense RNA, siRNA and miRNA are being experimentally applied as antisense therapy or to create knockout organisms to study gene function. For example, the suppression of protein synthesis by introducing antisense RNA, siRNA or miRNA into a cell may be useful to inhibit a number of infections or diseases in both plants and animals. A gene encoding the antisense RNA, siRNA or miRNA can be introduced fairly easily into organisms by using a plasmid vector or using a gene gun that shoots microscopic tungsten pellets coated with the gene into cells. Once the antisense RNA, siRNA or miRNA is introduced, it will specifically inhibit the synthesis of the target protein by binding to mRNA. Antisense RNA, siRNA or miRNA can be use in therapy, for example, for treating B-cell lymphomas and leukemias, treating HIV-1, cytomegalovirus, herpesvirus, asthma and cancers. Antisense RNA, siRNA or miRNA can also be used for commercial food production, for example, disease control and produce preservation. For example, siRNAs may be used as important tools for transcriptional modulating in silencing of mammalian genes by guiding DNA methylation.
Thus, an object of this invention is the identification of novel nucleotide analogues that can be used as antiviral or antitumor agents to inhibit diseases and conditions associated viruses and cancers.